Replication protein a binding transcriptional factor (RBT1) and uses thereof

ABSTRACT

The present invention relates to replication protein A (RPA) transcriptional factors. There is provided a nucleotide sequence encoding a replication protein A transcriptional activator 1 (RTB1) and a protein encoded by such a nucleotide sequence. RBT1 has a high activity and is highly transactivated in cancer cells. The sequence may be used to treat neoplastic disorders and in gene therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International ApplicationPCT/CA00/00948 filed Aug. 17, 2000 and designating the United States,now abandoned, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/149,472, filed Aug. 19, 1999; the entire contents of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to Replication Protein A (RPA) and moreparticularly to a RPA transcriptional factor to treat neoplasticdisorders such as cancer.

(b) Description of Prior Art

Replication Protein A (RPA), also known as replication factor A (RFA),is a ubiquitous and abundant heterotrimeric protein required for DNAreplication, repair and recombination in eukaryotes. RPA nonspecificallybinds single-stranded DNA and plays an essential role in the regulationof DNA metabolism via multiple protein interactions and/or RPAphosphorylation. More particularly, RPA binds single-stranded DNA withstrong affinity (association constant of 10⁹–10¹¹ M⁻¹) and greatestaffinity for polypyrimidine tracts. RPA also binds double-stranded DNAwith lower affinity and is likely to facilitate DNA unwinding. RPA mayplay a role in the regulation of transcription by binding regulatoryelements in promoters; in yeast, RPA binds specific regulatory sequencesin the promoters of DNA repair and metabolism genes (Singh K. et al.,1995, Proceedings of the National Academy of Science USA92(11):4907–11).

RPA is made of three subunits: a 70-kDa subunit (RPA70), a 32-kDa middlesubunit (RPA32) and a 14-kDa subunit (RPA14). The RPA32 subunit isphosphorylated in a cell cycle-dependent manner.

RPA-protein interactions appear to be largely mediated by the large70-kDa subunit (RPA70). RPA70 interacts with the p53, GAL4, VP16, EBNA1and SV40T antigens and with DNA polymerase alpha (Wold, M., 1997, AnnualReview of Biochemistry, “Replication Protein A: A Heterotrimeric,Single-Stranded DNA-binding Protein Required for Eukaryotic DNAMetabolism”). It is also important in interaction with DNA repairproteins involved in damage recognition and excision.

Interaction with XPF stimulates its 5′ junction-specific endonucleaseactivity, interaction with XPG targets this endonuclease to damaged DNA,and interaction with ERCC1 (ERCC1 also binds xeroderma pigmentosum groupA factor, XPA, which is another NER factor) promotes exonucleaseactivity.

The possibility of interaction by the aforementioned repair proteinswith RPA32 has not been clearly elucidated. However, interactions withsome proteins involved in DNA repair appear to be mediated by RPA32,such as interaction with XPA and uracil-DNA glycosylase. A region ofsignificant homology between uracil-DNA glycosylase and XPA was alsoreported, suggestive of the possibility of a common binding motif toRPA32 across several different proteins. Furthermore, some importantprotein interactions, such as with RAD52, appear to involve all threesubunits of RPA (Hays, S. et al., 1998, Molecular and Cellular Biology18(7):4400–4406).

In cells, RPA is phosphorylated by DNA-dependent protein kinase (DNA-PK)when RPA is bound to single-strand DNA, during the S phase and after DNAdamage; and also possibly by ATM.

Phosphorylation of RPA is observed in a cell-cycle dependent manner andin response to DNA damage (i.e. UV light, X-rays, cisplatin) ineukaryotic systems. This phosphorylation takes place predominantly onthe N-terminal region of RPA32 and was previously thought to be effectedby DNA-dependent protein kinase (DNA-PK). However, RPAhyperphosphorylation still takes place in SCID cells where DNA-PK isbelieved to be responsible for its repair and recombination defects.Ataxia telangiectasia mutated gene (ATM), an important cell cyclecheckpoint protein kinase belonging to the same kinase family as DNA-PK,may be responsible for the in vivo phosphorylation of RPA32. InSaccharomyces cerevisiae, the ATM homolog, MEC1, is essential for RPAphosphorylation. Furthermore, ionizing radiation-induced phosphorylationof RPA32 is deficient and reduced in primary fibroblasts from patientssuffering from ataxia telangiectasia in comparison to normal, agedfibroblasts.

The result of RPA32 phosphorylation on DNA metabolism is largelyunsolved. It has been noted that IR-induced RPA phosphorylation can beuncoupled from the S-phase checkpoint in ataxia telangiectasia cells,suggesting that RPA phosphorylation in itself is not necessary orsufficient for an S-phase arrest. Phosphorylation, however, may affectthe conformation of RPA, thereby modulating its affinity for DNA and itsprotein interactors, and altering the balance between DNA replicationand repair. Hyperphosphorylation of RPA32 in vivo is concordant with adecrease in the binding of RPA to single-stranded DNA. This observationis interesting to note since phosphorylated RPA32 is found predominantlyin the S-phase of the cell cycle.

RPA has been found to have a high affinity for UV-damaged andcisplatin-damaged DNA and the accompanying phosphorylated form of RPA iscorrelated strongly with a reduction of the in vitro DNA replicationactivity of the concerned cell extracts.

It would therefore be highly desirable to identify physiologicallyrelevant protein interactors of the RPA32 subunit of Replication ProteinA. Identification of such protein interactors would contribute to theunderstanding of DNA repair, transcription, and cell signaling. Theproteins involved in nucleotide excision repair (NER), for example, arequite numerous and the basis for their interaction and function is notyet completely understood. Understanding the regulation of thesepathways would assuredly lend insight into their role in cancersusceptibility. RPA, as a protein involved integrally in modulating DNArepair, replication and recombination, would be key to understanding theconnection between and within pathways. The implications to cancertherapeutics and/or prevention would be significant.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide a protein interactor ofthe RPA32 subunit of Replication Protein A (RPA).

Another aim of the present invention is to provide a RPA transcriptionalfactor to treat neoplastic disorders such as cancer.

In accordance with the present invention, there is provided a genehaving the characteristics of a gene encoded by a nucleotide sequence asset forth in FIG. 1 (SEQ ID NO:1).

The gene may be from a human, a mouse, a rat or a yeast.

In accordance with the present invention, there is also provided aprotein having the identifying characteristics of a protein encoded by anucleotide sequence as set forth in FIG. 1 (SEQ ID NO:1).

The protein may be from a human, a mouse, a rat or a yeast.

Antibodies may be raised against the gene.

The gene, replication protein A binding transcriptional activator 1(RBT1), encodes a protein interactor of the Replication Protein A (RPA)More particularly, a protein interactor of the Replication Protein A32KD subunit was identified. RBT1 binds RPA32.

The RBT1 gene has a high activity in cancer cells compared to normalcells, may be involved in carcinogenesis and is highly transactivated incancer cells.

The RBT1 nucleotide and/or amino acid sequences may be used to generatereagents, such as plasmids, antibodies and inhibitors, includingantisense/antibodies which may be used in treating neoplastic disorderssuch as cancer.

The RBT1 sequence of the present invention may also be used for thepreparation of a medicament for gene therapy, wherein he RBT1 sequenceis used as a specific promoter to overexpress genes of interest inspecific tissues.

In accordance with another embodiment of the present invention, there isprovided a method of gene therapy, which comprises the use of RBT1sequence as a promoter for overexpressing a gene in a suitable tissue.

The RBT1 gene may further be used to induce apoptosis in cells such ascancerous cells, by modulating its expression using molecular orchemical approaches.

The RBT1 sequence of the present invention may also be used to developantisenses and/or inhibitors to treat diseases including cancers andleukemia.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates the nucleotide (SEQ ID NO:1) and the amino acidsequence (SEQ ID NO:2) of RBT1.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided a genesequence encoding a protein interactor of Replication Protein A,identified using the yeast two-hybrid system. The gene, named RPABinding Transcriptional Activator 1 (RBT1), has a putative open readingframe of 196 amino acids. The coding sequence of RBT1 corresponds toseveral expressed sequence tags (ESTs), including one derived from anovary tumor cell line. The gene of the present invention acts as astrong transcriptional activator in yeast and mammalian cells.Furthermore, transcriptional activation, as assayed by a luciferasereporter gene, demonstrated that the activity of the RBT1 gene of thepresent invention is higher in cancer cells compared to normalnon-immortalized cells. RBT1 expression is higher in cancer cellscompared to normal cells. More particularly, a protein interactor ofHuman Replication Protein A 32 (RPA32) was identified.

BLASTP homology searches against the deduced amino acid sequence of RBT1reveal that it is an undefined protein with little homology to knownprotein sequences. Further, BLASTN homology searches only identifiedapproximately 20 human expressed sequence tags (ESTS) which had highhomology to RBT1.

Northern blot using an RBT1 DNA probe showed one transcript ofapproximately 1.55 kb in size. In silicio analysis suggested that RBT1consists of an open reading frame (ORF) of 196 amino acids and atheorectical molecular weight of 22 kDa. This is in agreement withWestern Blot analysis.

Differential expression of RBT1 was also investigated as it relates tocancer. Semi-quantitative analysis has shown that RBT1 is at least tentimes more expressed in cell line H661 (cancer cells) than NHBEC (normalcells).

Various cell lines are investigated to ascertain whether RBT1 hasrelevance to carcinogenesis. In silicio analysis also suggests that theN-terminal domain of RBT1 contains a putative DNA binding domain.Whether RBT1 binds specific DNA regulatory elements is also beinginvestigated.

The presence of an acidic domain in the C-terminal domain of RBT1 led toinvestigate whether RBT1 was a potential transcriptional activator.RBT1, fused to the LexA binding domain, strongly promotes transcriptionof reporter genes LacZ and HIS3 in the yeast two-hybrid system,suggesting its possible role as a transcriptional activator.

RBT1 deletion constructs were designed to determine the transactivatingdomain, and to define the domain which is essential for RPA32interaction. The transactivation domain of RBT1 resides within 30 aminoacids at the C-terminal. Truncation of RBT1 from the C-terminal endresults in significant reduction of transactivation of the reportergenes.

A mammalian transactivation assay confirmed that a GAL4-RBT1 fusionprotein indeed acts as a strong transcriptional activator. Furthermore,transcriptional activation, as assayed by a luciferase reporter gene,although high in all cancer cell lines examined, is at least 4 timeshigher in cell line MCF7. Transactivation studies were also performedusing a mammalian system to verify that RBT1 acts as a transcriptionalactivator in its native cellular environment. RBT1, fused to a GAL4 DNAbinding domain, strongly promotes transcription of of B-gal activitywith just 60 bp deleted from the 3′, suggesting that the potentialtranscriptional activation domain of RBT1 lies at the carboxy terminal.

Similar constructs may be cloned into a vector for transfection intohuman cells, using an in-frame fusion to GAL4 DNA-binding domain andutilizing a second plasmid bearing a luciferase reporter gene under thecontrol of several GAL4 binding sites. These experiments determinewhether the transactivation found in the yeast system arephysiologicallly relevant.

RBT1 may be overexpressed in various human cell lines to ascertainpossible phenotypic effects. Experiments may include UV and chemicalchallenge.

Antibodies against RBT1 may be raised for subsequent proteinlocalization experiments in human cells. This antibody may also be usedfor various co-immunoprecipitation experiments to show RPA-RBT1 binding.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

1. An isolated nucleotide sequence encoding a protein having the aminoacid sequence as set forth in SEQ ID NO:
 2. 2. An isolated proteinhaving the amino acid sequence as set forth in SEQ ID NO:
 2. 3. A methodfor increasing the in vitro transcription of a gene, the methodcomprising introducing the gene operably linked to the isolatednucleotide sequence according to claim 1 into a cell in vitro,expressing the sequence to produce a transcriptional activator protein,thereby increasing the transcription of the gene.
 4. The isolated orrecombinant nucleotide sequence according to claim 1, said isolatednucleotide sequence having the nucleotide sequence as set forth in SEQID NO: 1.